User manual LEICA TCS SMD DATASHEET

[. . . ] Leica TCS SMD Series Single Molecule Detection Platform for Obtaining Meaningful, Reliable Results: FCS, FCCS, FLIM, FRET, FLCS, Gated FCS Protein diffusion in polymer matrix 1, 0 normalized autocorrelation G() 0, 8 0, 6 Long polymers: Normal diffusion 0, 4 Short polymers: Sub-diffusion 0, 2 0, 0 1 2 0, 01 0, 1 1 10 100 Lag time [ms] 3 · All in one ­ a single platform to study the dynamics of life · Global control by full system integration · Quantification with maximum content · Dedicated application wizards for fast, reproducible work · Immediate results by intelligent hardware and software presets Leica Design by Christophe Apothéloz 2 4 Molecular interactions, such as protein complex formation, protein-DNA or ligand-receptor binding are extremely significant for modern biology (1, 2). The specific identification of interaction partners and quantification of binding parameters are crucial for the understanding the biological, chemical, and physical processes in live cells. This information is necessary for goal-oriented development of agents that influence biochemical reactions on a molecular level, which is essential in pharmaceutical drug design and biomedical research. Leica TCS SMD Series Quantify Life! Single Molecule Detection (SMD) and analysis is an elegant way to examine dynamics and interactions inside cellular systems. This includes the quantitative characterization of biochemical reaction kinetics and equilibrium on a molecular level. [. . . ] This gives the freedom and flexibility to choose the spectral detection range for FLIM. 3 1 2 SMD FLIM wizard: complex experiments easily run Using the SMD FLIM wizard the user defines all relevant parameters for FLIM acquisition, such as scan speed and format or acquisition time of single FLIM images. These settings are transferred to and automatically used by SymPhoTime Software. The SMD FLIM wizard offers a variety of FLIM scan modes to set up more complex sequences of FLIM data acquisition : FLIM volume stacks give information about the lifetime distribution in tissues or small organisms. With FLIM time series the researcher can follow dynamic changes of fluorescence lifetimes, especially in live cells or tissues. Volume stacks and time series can be easily combined. A new dimension of knowledge ­ FLIM lambda stacks: SP FLIM detectors are used for automated acquisition of FLIM lambda stacks, i. e. , FLIM image series at defined bands of the emission spectrum. Lifetime emission spectra are especially useful for characterization and identification of autofluorescence or new chromophores, for better separation of dyes with similar properties, and for identification of conformational states and aggregation of chromophores. Automated brightness control provides maximum data reliability: this unique feature generates images with pre-defined brightness. It ensures that the photon statistics of all recorded images are consistent. Automated brightness control can be used for volume and lambda stacks. Intrinsically it compensates for a fluorescence intensity decrease caused by photo-bleaching or light absorbance in deeper sample sections. FLIM 2 FLIM Advantages · Immune to effects such as: ­ Concentration fluctuations caused by diffusion, or photo-bleaching (without photo-conversion) ­ Shading in thick samples ­ Fluctuations of excitation intensity ­ Light source noise · Internally calibrated FRET · Unperturbed conditions in unstained samples 14 3D reconstruction of FLIM z-stack Complementary technologies available in LAS AF · FRET SE Wizard · FRET AB Wizard 16 Host ­ Pathogen Interaction ­ Identification of Invading Hyphae in Tomato Fruit Intensity Image [Cnts] 700 0 Average Lifetime [ns] 2, 97 0, 4 Chloroplasts Fungal hyphae Putative parenchyma 15 1. FLIM stack Intensity spectra 1, 2 ROI 1 ROI 2 ROI 3 Average lifetime spectra 2, 5 ROI 1 ROI 2 ROI 3 Normalized fluorescence intensity 1, 0 2, 0 0, 8 Fluorescence lifetime [ns] 440 490 540 590 640 690 740 0, 6 1, 5 0, 4 1, 0 0, 2 0, 5 0, 0 0, 0 440 490 540 590 640 690 740 Wavelength [nm] wavelength [nm] 3. Fluorescence intensity spectra ROI1 - Fluorescence intensity vs. Spectra of average lifetimes ROI2 - Fluorescence intensity vs. lifetime and wavelength ROI3 - Fluorescence intensity vs. lifetime and wavelength 3, 0e+5 2, 5e+5 1, 4e+5 1, 2e+5 2, 5e+5 Fluorescence intensity [counts] Fluorescence intensity [counts] 2, 0e+5 1, 5e+5 1, 0e+5 5, 0e+4 0, 0 -5, 0e+4 -1, 0e+5 1, 0e+5 8, 0e+4 6, 0e+4 4, 0e+4 2, 0e+4 0, 0 -2, 0e+4 -4, 0e+4 Fluorescence intensity [counts] -1, 0e+5 -5, 0e+4 0, 0 5, 0e+4 1, 0e+5 1, 5e+5 2, 0e+5 2, 5e+5 3, 0e+5 -4, 0e+4 -2, 0e+4 0, 0 2, 0e+4 4, 0e+4 6, 0e+4 8, 0e+4 1, 0e+5 1, 2e+5 1, 4e+5 2, 0e+5 1, 5e+5 1, 0e+5 -5, 0e+4 0, 0 5, 0e+4 1, 0e+5 1, 5e+5 2, 0e+5 2, 5e+5 5, 0e+4 0, 0 -5, 0e+4 740 life 1, 0 1, 5 tim e 2, 0 [ns 2, 5 ] 3, 0 490 440 540 590 640 690 740 th leng Wave ] [nm life 1 tim 2 e [ns ] 3 4 490 440 540 590 640 690 Wave th leng ] [nm life 1, 0 1, 5 tim e 2, 0 [ns 2, 5 ] 0, 5 3, 0 490 440 540 590 640 690 740 Wave th leng [nm ] 5. Spectral lifetime fingerprinting Note: Data visualization using external software 17 FLIM-FRET: The Molecular Ruler A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well. In a FRET experiment the potential binding partners are labeled with spectrally distinct fluorophores in such a way that the emission spectrum of the donor molecule overlaps the excitation spectrum of the acceptor molecule. If both interaction partners are in close contact at a distance of only a few nanometers, the excited donor can transfer its energy to the acceptor. In turn, the acceptor emits a fluorescence photon and the fluorescence lifetime of the donor molecule decreases. [. . . ] Improved signalto-noise ratio evidenced by larger amplitude in FLCS mode (blue curve) compared to standard FCS (red curve). Volker Buschmann, PicoQuant GmbH, Berlin for experimental support. GalNacT2-GFP expressed in HeLa cells analyzed with FLCS. Fluorescence image using SPAD detectors (yellow) with DIC overlay (1). [. . . ]